We plan to solve the solution structure of MMOB, a small 16.5 kDa enzyme that regulates electron and oxygen transfer reactions in the methane monooxygenase system. Using our current expression and purification methods, yields of -18 mg of pure, homogeneous protein per liter of M-9 culture medium are obtained. The purified MMOB protein is stable at 25 OC and can be concentrated in excess of 3 mM and freeze dried without significant loss of activity. With the help of Dr. Chris Turner, we have recorded high resolution one dimensional, and two dimensional (NOESY and TOCSY) spectra using the FBML 750 MHz spectrometer which suggest that MMOB is an excellent candidate for NMR structural investigation. The determination of the MMOB structure will involve making sequential resonance assignments using homonuclear (H) and heteronuclear (H- 15 N) TOCSY and NOESY data. Two and three dimensional TOCSY experiments will be used to identify amino acid spin systems. Uniformly labeled "C-"N - MMOB will be used in CBCA(CO)NH and HNCACB studies to complete the assignment of the primary amino acid sequence. Experimentally determined distance and dihedral angle constraints will be used in molecular dynamics calculations and simulated annealing refinement to solve the MMOB structure. The high field 750 and 600 MHz instruments at the FBML and guidance from Dr. Turner in the development of the appropriate multidimensional homonuclear and heteronuclear experiments are essential to the success of this investigation.